This invention relates generally to modified oligonucleotides of preselected sequence, and more specifically to single-stranded oligonucleotides including nucleotides modified for the attachment of detectable reporter groups or solid support.
Nucleic acids, which are the carriers of genetic information between generations, are composed of linearly arranged individual units called nucleotides. Each nucleotide has a sugar phosphate group to which is attached one of the pyrimidine or purine bases, adenine (A), thymine (T), uracil (U), guanine (G) or cytosine (C). In the native state, single-stranded nucleic acids form a double helix through highly specific bonding between bases on the two strands; A will bond only with T or U, G will bond only with C. Thus a double stranded nucleic acid will form where, and only where, the sequence of bases in the two strands is complementary.
The understanding of complementary bonding between nucleic acids permits a variety of applications. For example, labelled nucleic acids of known base sequence, termed genetic probes, may be used to detect the presence of complementary nucleic acid in a sample. Such technology provides the most sensitive method available for determining the existence of a particular gene in a cell or of organisms such as viruses and bacteria. The ideal genetic probe would be of uniform length to allow predictable hybridization behavior and would be of homogeneous sequence to minimize cross reactivity with non-targeted nucleic acids. Moreover, it would be single-stranded and easily detectable.
One factor which has limited the utilization of genetic probes has been the difficulties encountered in producing detectable single-stranded nucleic acids having a preselected sequence. Two techniques of nucleic acid synthesis are currently used: enzymatic and chemical, i.e. non-enzymatic. The enzymatic synthesis of nucleic acid requires preexisting DNA for a template and utilizes natural cellular enzymatic mechanisms to facilitate replication of DNA segments. Two conventional examples of such synthesis are the nick translation protocol (Rigby et al., 1977 J. Mol. Biol. 113:237-251) and the gap-filling reaction (Bourguignon et al., 1976. J. Virol. 20:290-306). In both methods, preexisting DNA is contacted with an enzyme known as a DNase which nicks the strand, causing a break between a 3'-hydroxyl group and the adjacent 5'-phosphate. Such nicked or gapped DNA then serves as both a template and a primer. In the presence of a DNA polymerase, such as POL I which is isolated from E. coli, free nucleotides are successively condensed on to the 3' hydroxyl group while nucleotides adjacent to the 5' end of the nick are simultaneously cleaved. Double-stranded DNA having new strands composed of the added nucleotides is thus formed. Although DNA polymerases are the enzymes most commonly used in such procedures, other enzymes such as terminal transferases, reverse transcriptases and RNA polymerases can also be used with similar results.
If one or more of the provided nucleotides are modified, for example to include a label, such modifications will be incorporated into the new strand. Only a limited array of modifications may be utilized in such a method, however, due to the interference of the modifications with the activity of the enzymes. Radioisotopes, such as .sup.32 P or .sup.14 C, may be readily incorporated since they closely resemble the natural isotopes, and thus radioactively labelled probes have been widely used. Because of the potential hazards associated with handling and disposing of radioactive materials and their inherent instability, however, radioactive probes are undesireable.
Certain other modified bases have been incorporated into oligonucleotides prepared by enzymatic synthesis. Ward et al., European Patent Application No. 82301804.9 disclose pyrimidine and purine bases having certain moieties attached, such as biotin, which are capable of complexing with a polypeptide for detection. These modified bases can be incorporated into enzymatically produced nucleic acids. However, hybridization probes produced by such methods have inherent drawbacks which limit their usefulness. For example, enzymatic synthesis relies on nicked preexisting DNA to serve as a template. Because multiple nicks are introduced randomly in a single chain by contact with a DNase, double-stranded oligonucleotides of widely different composition, sequence, and length will be simultaneously produced. Length of product chains vary considerably, usually from 400 to 1000 bases in length. In general, chains of over 200 bases are termed "polynucleotides" while those under 200 bases are termed "oligonucleotides." Such enzymatic synthesis chains may reach a length of several thousand nucleotides but for practical purposes, they generally cannot be made less than about two hundred nucleotides in length. The absolute length cannot be controlled, however, and the product will be a heterologous mixture of lengths and sequences. No conventional method permits separation and purification of these heterogeneous pieces. Moreover, it is not possible to control the site at which the modified nucleotide is incorporated into the newly formed chain. While the identity of the particular nucleotide which is modified does determine that the label will be incorporated opposite a position of the complementary nucleotide in the template, the method does not permit the synthesis of a polynucleotide having modifications at particular preselected sites among those available. More importantly, the range of modifications of the nucleotides which can be incorporated is limited to those which will be recognized and incorporated by the enzymes.
To a limited extent, modifications to nucleic acids have also been introduced by post-synthetic modification of an enzymatically synthesized nucleic acid, such as by mercuration or palladium catalyzed addition reactions. Only cytosine residues are susceptible to such addition reactions, however; thymine and purine bases cannot be modified by this method. Moreover, as with enzymatic incorporation of modified bases, the particular site at which the modification may be introduced can not be preselected and both strands are randomly modified. Where the stoichiometry of the reaction is controlled so as to modify only a limited proportion of the available nucleotides, the modifications will be introduced randomly at sites appropriate to cytosine incorporation, thereby producing a heterogeneous population of modified nucleic acids; see Bigge, et al., (1981). J. Carb. Nucleosides Nucleotide, 8:259 (1981). Furthermore, it has been demonstrated that where the reaction conditions are intensified so as to modify substantially all available nucleotides, undesired chemical degradation of the oligonucleotide ensues. Dale et al., (1975). Biochem. 14:2447. This method has not been used to incorporate labels or reporter groups.
The prior art methods of enzymatic synthesis require double-stranded DNA as a template, and produce double-stranded nucleic acids having label incorporated in both strands. Moreover, the resulting nucleic acids are heterogenous, varying both in sequence, length, and in position of the modified bases. Enzymatic synthesis cannot produce a single stranded probe of preselected length, preselected sequence having unique reporter groups defined by site and number. Furthermore, the scope of modifications obtainable in the oligonucleotide product is severely restricted as the enzymes required for modification can only recognize and incorporate a very limited array of modified nucleotides in both strands of a double-stranded, nonuniform nucleic acid. As a result proteins, nucleic acids, carbohydrates, fluorophors, and lumiphors cannot be incorporated as labels by these methods.
Naturally occurring nucleotides may be condensed into single-stranded oligonucleotides of preselected sequence and length using chemical, or non-enzymatic, methods of synthesis. Such methods have been reviewed by Matteucci, et al., (1982). J. Amer. Chem. Soc. 103:3185. Chemical synthesis usually involves successive coupling of an activated nucleotide monomer and a free hydroxyl-bearing terminal unit of a growing nucleotide chain. The coupling is effected through a reactive phosphorous-containing group, such as a phosphate diester or more often, a phosphite triester. Phosphochloridite (Letsinger, et al., (1980). J. Org. Chem. 45:2715) and phosphoamidite (Caruthers, et al., U.S. Pat. No. 4,458,066) reactions are commonly used. Caruthers teaches the synthesis of oligonucleotides containing as many as 30 bases composed of only naturally-occurring nucleotides. However, no chemical synthesis of oligonucleotides incorporating modified bases or reporter groups of any type has been disclosed in the prior art.
Accordingly, there exists a long felt and compelling need for single-stranded oligonucleotides of preselected sequence and length having incorporated therein modified nucleotides capable of detection. Such modifications should be non-radioactive and preferably allow accurate and inexpensive detection. The present invention satisfies this need and provides related advantages as well.